ABSTRACT: Ribonucleic acid (RNA), essential for protein production and immune function, undergoes glycosylation, a process that attaches glycans to RNA, generating unique glycoRNAs. These glycan-coated RNA molecules regulate immune responses and may be related to immune disorders. However, studying them is challenging due to RNA's fragility. Therefore, a robust method for identifying glycoRNA is important. To address this, we optimized parameters for RNA stability, oxidation, and digestion, thereby enriching and identifying glycoRNAs. This breakthrough paves the way for exploring their potential interactions with immune receptors and tumor suppression. Our approach involved investigating factors such as preservation reagent, enzyme buffer, digestion temperature, oxidant, glycosidase, and incubation time. We successfully optimized digestion conditions, achieving efficient cleavage of both N-linked and O linked glycoRNAs at room temperature using 25 mM ammonium bicarbonate, demonstrating the effectiveness of this method. Additionally, RNA preservation in RNAlater at -80°C allows controlled release of glycoRNAs within hours. While sequential digestion of different glycoRNA types is possible, significant degradation occurs after the first glycosidase step. Thus, we recommend separate harvesting for each type of glycoRNA. These optimized protocols, utilizing SPCgRNA and TnORNA methods, pave the way for further research on N- and O-glycoRNAs in health and disease.
